These techniques have been widely adopted by research groups involved in microbial cell biology, and the plasmid vectors have now been distributed directly to well over 100 laboratories world-wide, although they are now also distributed by the Bacillus Genetic Stock Centre. These techniques were instrumental in determining the establishment of compartment specificity of developmentally regulated s-factors during sporulation in B. subtilis (Lewis et al., PNAS 91, 3849-3853 (1994)). I also showed that prespore specific accumulation of the transcription regulator SpoIIAA was responsible for initiation of compartment-specific gene expression during development, that this accumulation was probably due to prespore-specific activity of the phosphatase SpoIIE, and that a programme of proteolysis was initiated following this activation event (Lewis et al., Genes Cell 1, 881-894 (1996); Lewis et al., J. Bacteriol 180, 3276-3284 (1998)). I was also involved in work that showed that the highly conserved SpoIIIE (FtsK) protein is a DNA translocase that moves DNA through a division septum (Wu et al., Genes Dev 9, 1316-1326 (1995)). This was the first example of such a phenomenon, and was a very significant finding as previously it was assumed that DNA was segregated into daughter cells/different compartments prior to division septum formation. Finally, I have shown that transcription and translation are spatially separated within bacteria (Lewis et al., EMBO J 19, 710-718 (2000)). This was an unexpected result as the 2 processes were thought to be very tightly coupled in bacteria. Furthermore, transcription becomes concentrated into a sub-fraction of the bacterial nucleoid at higher growth rates. These transcription foci have been shown to be the sites of rRNA synthesis, and my laboratory is now focussing on characterising transcription complexes and their structure in detail.

Research ExpertiseMy research career has focused on the gram positive bacterium Bacillus subtilis. During my PhD I purified and characterised the replication terminator protein (RTP) and its DNA binding sites (Lewis et al., J. Bacteriol 171, 3564-3567 (1989); Lewis et al., J. Mol. Biol. 214, 73-84 (1990)). These sites are regions of DNA where oppositely moving replication forks meet and newly replicated chromosomes are resolved prior to segregation. During my post-doctoral research I was responsible for the development of cell biological techniques and vectors for visualising gene expression and protein localisation in live bacterial cells (Lewis et al., PNAS 91, 3849-3853 (1994); Lewis et al., Mol Microbiol 13, 655-662 (1994); Lewis and Errington, Microbiology 142, 733-740 (1996); Lewis and Marston, Gene 227, 101-109 (1999); Feucht and Lewis, Gene 264, 289-297 (2001)). These techniques have been widely adopted by research groups involved in microbial cell biology, and the plasmid vectors have now been distributed directly to well over 100 laboratories world-wide, although they are now also distributed by the Bacillus Genetic Stock Centre. These techniques were instrumental in determining the establishment of compartment specificity of developmentally regulated s-factors during sporulation in B. subtilis (Lewis et al., PNAS 91, 3849-3853 (1994)). I also showed that prespore specific accumulation of the transcription regulator SpoIIAA was responsible for initiation of compartment-specific gene expression during development, that this accumulation was probably due to prespore-specific activity of the phosphatase SpoIIE, and that a programme of proteolysis was initiated following this activation event (Lewis et al., Genes Cell 1, 881-894 (1996); Lewis et al., J. Bacteriol 180, 3276-3284 (1998)). I was also involved in work that showed that the highly conserved SpoIIIE (FtsK) protein is a DNA translocase that moves DNA through a division septum (Wu et al., Genes Dev 9, 1316-1326 (1995)). This was the first example of such a phenomenon, and was a very significant finding as previously it was assumed that DNA was segregated into daughter cells/different compartments prior to division septum formation. Finally, I have shown that transcription and translation are spatially separated within bacteria (Lewis et al., EMBO J 19, 710-718 (2000)). This was an unexpected result as the 2 processes were thought to be very tightly coupled in bacteria. Furthermore, transcription becomes concentrated into a sub-fraction of the bacterial nucleoid at higher growth rates. These transcription foci have been shown to be the sites of rRNA synthesis, and my laboratory is now focussing on characterising transcription complexes and their structure in detail.

Invitations

Participant

ReviewerOrganisation: Various Publishers in the field of Environmental and Life Science
Description:
Have been invited to write reviews for top journals in field (Molecular Microbiology, Microbiology and international Review of Cytology) and 2 book chapters in the last 3 years. Work is also now appearing in Microbiology text books.

Our ongoing research focused on targeting transcription initiation in bacteria has resulted in synthesis of several classes of mono-indole and mono-benzofuran inhibitors that targ... [more]

Our ongoing research focused on targeting transcription initiation in bacteria has resulted in synthesis of several classes of mono-indole and mono-benzofuran inhibitors that targeted the essential protein-protein interaction between RNA polymerase core and s 70/s A factors in bacteria. In this study, the reaction of indole-2-, indole-3-, indole-7- and benzofuran-2-glyoxyloyl chlorides with amines and hydrazines afforded a variety of glyoxyloylamides and glyoxyloylhydrazides. Similarly, condensation of 2- and 7-trichloroacetylindoles with amines and hydrazines delivered amides and hydrazides. The novel molecules were found to inhibit the RNA polymerase-s 70/s A interaction as measured by ELISA, and also inhibited the growth of both Gram-positive and Gram-negative bacteria in culture. Structure-activity relationship (SAR) studies of the mono-indole and mono-benzofuran inhibitors suggested that the hydrophilic-hydrophobic balance is an important determinant of biological activity.

Our ongoing research focused on targeting transcription initiation in bacteria has resulted in synthesis of several classes of mono-indole and mono-benzofuran inhibitors that targ... [more]

Our ongoing research focused on targeting transcription initiation in bacteria has resulted in synthesis of several classes of mono-indole and mono-benzofuran inhibitors that targeted the essential protein-protein interaction between RNA polymerase core and s70/sA factors in bacteria. In this study, the reaction of indole-2-, indole-3-, indole-7- and benzofuran-2-glyoxyloyl chlorides with amines and hydrazines afforded a variety of glyoxyloylamides and glyoxyloylhydrazides. Similarly, condensation of 2- and 7-trichloroacetylindoles with amines and hydrazines delivered amides and hydrazides. The novel molecules were found to inhibit the RNA polymerase-s70/sA interaction as measured by ELISA, and also inhibited the growth of both Gram-positive and Gram-negative bacteria in culture. Structure-activity relationship (SAR) studies of the mono-indole and mono-benzofuran inhibitors suggested that the hydrophilic-hydrophobic balance is an important determinant of biological activity.

Triterpenoid saponins are involved in plant defense systems to inhibit bacterial invasion. A new series of hydrophilic ocotillol-type triterpenoid derivatives 5-26 have been synth... [more]

Triterpenoid saponins are involved in plant defense systems to inhibit bacterial invasion. A new series of hydrophilic ocotillol-type triterpenoid derivatives 5-26 have been synthesized with antibacterial activity against Gram-positive bacteria, including a community associated methicillin-resistant Staphylococcus aureus (CA-MRSA; strain USA300). From this series, compounds 6 and 15 were found to be the most active, both with MIC values of 2 Âµg/mL against B. subtilis 168 and 8 Âµg/mL against S. aureus USA300, respectively. Furthermore, subsequent assays showed that compounds 6 and 15 displayed strong synergistic effects at sub-MIC levels against both S. aureus USA300 and B. subtilis 168 when combined with two commercial antibiotics, kanamycin and chloramphenicol. Preliminary structure-activity relationship studies were also performed.

Acinetobacter species are widely distributed bacteria in the environment, and have recently gained notoriety as opportunistic nosocomial pathogens. Here we characterize a novel RN... [more]

Acinetobacter species are widely distributed bacteria in the environment, and have recently gained notoriety as opportunistic nosocomial pathogens. Here we characterize a novel RNA polymerase-interacting protein named acidic transcription factor A, AtfA. It is small and highly acidic, and is widely distributed throughout the Â¿ proteobacteria, including other significant pathogens in the genera Moraxella, Pseudomonas, Legionella and Vibrio. In the model species A. baylyiADP1, deletion of atfA significantly affects expression of over 500 genes, resulting in a large cell phenotype, reduced cell fitness, impaired biofilm formation and twitching motility, and increased sensitivity to antibiotics. Deletion of atfA also causes dramatically enhanced sensitivity to ethanol, which is an important growth promoter and virulence factor in Acinetobacter spp. The results suggest that auxiliary factors of RNA polymerase with important biological roles remain to be discovered.

We describe a dual vector-based system for overproduction of recombinant Escherichia coli RNA polymerase (RNAP). A cleavable deca-histidine tag (His10) was incorporated into the C... [more]

We describe a dual vector-based system for overproduction of recombinant Escherichia coli RNA polymerase (RNAP). A cleavable deca-histidine tag (His10) was incorporated into the C-terminus of the Ã' subunit to facilitate protein purification. Unique restriction sites were introduced into the genes encoding the Ã and Ã' subunits (rpoB and rpoC, respectively), facilitating mutation of functionally significant subunit fragments through insertion of modified PCR fragments into the appropriate vector. RNAP with an R275A substitution in the Ã' subunit, which is essential for interaction with transcription initiation factor s, was generated and exhibited reduced activity compared to native recombinant RNAP.

Acinetobacter species are widely distributed bacteria in the environment, and have recently gained notoriety as opportunistic nosocomial pathogens. Here we characterize a novel RN... [more]

Acinetobacter species are widely distributed bacteria in the environment, and have recently gained notoriety as opportunistic nosocomial pathogens. Here we characterize a novel RNA polymerase-interacting protein named acidic transcription factor A, AtfA. It is small and highly acidic, and is widely distributed throughout the Â¿ proteobacteria, including other significant pathogens in the genera Moraxella, Pseudomonas, Legionella and Vibrio. In the model species A. baylyiADP1, deletion of atfA significantly affects expression of over 500 genes, resulting in a large cell phenotype, reduced cell fitness, impaired biofilm formation and twitching motility, and increased sensitivity to antibiotics. Deletion of atfA also causes dramatically enhanced sensitivity to ethanol, which is an important growth promoter and virulence factor in Acinetobacter spp. The results suggest that auxiliary factors of RNA polymerase with important biological roles remain to be discovered.

We have examined the localization of DNA replication of the Bacillus subtilis phage f29 by immunofluorescence. To determine where phage replication was localized within infected c... [more]

We have examined the localization of DNA replication of the Bacillus subtilis phage f29 by immunofluorescence. To determine where phage replication was localized within infected cells, we examined the distribution of phage replication proteins and the sites of incorporation of nucleotide analogues into phage DNA. On initiation of replication, the phage DNA localized to a single focus within the cell, nearly always towards one end of the host cell nucleoid. At later stages of the infection cycle, phage replication was found to have redistributed to multiple sites around the periphery of the nucleoid, just under the cell membrane. Towards the end of the cycle, phage DNA was once again redistributed to become located within the bulk of the nucleoid. Efficient redistribution of replicating phage DNA from the initial replication site to various sites surrounding the nucleoid was found to be dependent on the phage protein p16.7.

Lewis PJ, Harry E, 'Cellular targeting of proteins involved in positioning the division site in bacteria', Proceedings of the Australian Society for Biochemistry and Molecular Biology, Sydney, NSW (2002) [E3]

Research Supervision

Current Supervision

Commenced

Research Title / Program / Supervisor Type

2015

Elucidating the Function of the Novel Transcription Factor AtfA and its Relevance to Virulence and Persistence in the y ProteobacteriaBiological Sciences, Faculty of Science and Information TechnologyPrincipal Supervisor

2013

Understanding Chromosome Dimer Resolution Systems of a Pathogenic Bacteria at a Molecular LevelBiological Sciences, Faculty of Science and Information TechnologyCo-Supervisor

2012

Development of Genetic Tools for the Quantitative Assessment of Gene Depletion in Pathogenic BacteriaBiological Sciences, Faculty of Science and Information TechnologyCo-Supervisor

Structural analysis of RNA polymeraseMicrobiology, University of NewcastleSole Supervisor

Past Supervision

Year

Research Title / Program / Supervisor Type

2014

Increased Understanding of the Molecular Interactions Involved in Bacterial Transcription and RecombinationBiological Sciences, Faculty of Science and Information TechnologyPrincipal Supervisor

2013

Investigation of the Transcription Complex in Acinetobacter baylyi ADP1 and the Identification of the Novel Small Acidic Transcription Factor AtfABiological Sciences, Faculty of Science and Information TechnologyPrincipal Supervisor

2013

Genetic Regulation of Embryo Development and Formation of Seed Storage Products in the Legume Model Medicago truncatulaBiological Sciences, Faculty of Science and Information TechnologyCo-Supervisor

2012

The Characterisation of the Interaction between PcrA and RNA PolymeraseBiological Sciences, Faculty of Science and Information TechnologySole Supervisor

2011

The RNA Polymerase-Q Interaction as a Target for Potential Novel AntimicrobialsBiological Sciences, Faculty of Science and Information TechnologyPrincipal Supervisor

2010

The Structure of Bacterial RNA Polymerase in Complex With the Essential Transcription Elongation Factor NusABiological Sciences, Faculty of Science and Information TechnologySole Supervisor

2008

The Composition of Transcription Complexes in Bacillus SubtilisBiological Sciences, Faculty of Science and Information TechnologySole Supervisor

2007

Interaction of C-terminus of PcrA with the B subunit of RNA polymeraseMicrobiology, University of NewcastleSole Supervisor

2006

Characterisation of the interaction between PcrA and the B-subunit of RNA polymeraseMicrobiology, University of NewcastleSole Supervisor

2005

Development of an engineered Bacillus subtilis strain for the detection of bioavailable environmental toxicantsMicrobiology, University of NewcastleSole Supervisor

2005

Characterisation of the interaction between PcrA and the beta subunit of RNA polymeraseMicrobiology, University of NewcastleSole Supervisor

2005

Interaction of transcription factors NusB and NusE in Bacillus subtilisMicrobiology, University of NewcastleSole Supervisor

2005

The design and testing of inhibitors of interactions between essential transcription factors and RNA polymeraseMicrobiology, University of NewcastleSole Supervisor

2003

Interactions between RNA polymerase and transcription factors: An integrated approachMicrobiology, University of NewcastleSole Supervisor

2003

Subcellular localisation of the transcription elongation factors NusB, NusG and GreA in Bacillus subtilisMicrobiology, University of NewcastleSole Supervisor

2003

Analysis of translation elongation factors EF-Tu and EF-G in Bacillus subtilisMicrobiology, University of NewcastleSole Supervisor

2003

Analysis of translation initiation factors in Bacillus subtilisMicrobiology, University of NewcastleSole Supervisor

2002

Subcellular localisation of tranlsation factors in Bacillus subtilisMicrobiology, University of NewcastleSole Supervisor

2001

Subcellular localisation of ATP synthase in Bacillus subtilisMicrobiology, University of NewcastleSole Supervisor

2001

Subcellular localisation of NusA in Bacillus subtilisMicrobiology, University of NewcastleSole Supervisor